Ubiquitin- and ATP-dependent unfoldase activity of P97/VCP•NPLOC4•UFD1L is enhanced by a mutation that causes multisystem proteinopathy

Proc Natl Acad Sci U S A. 2017 May 30;114(22):E4380-E4388. doi: 10.1073/pnas.1706205114. Epub 2017 May 16.

Abstract

p97 is a "segregase" that plays a key role in numerous ubiquitin (Ub)-dependent pathways such as ER-associated degradation. It has been hypothesized that p97 extracts proteins from membranes or macromolecular complexes to enable their proteasomal degradation; however, the complex nature of p97 substrates has made it difficult to directly observe the fundamental basis for this activity. To address this issue, we developed a soluble p97 substrate-Ub-GFP modified with K48-linked ubiquitin chains-for in vitro p97 activity assays. We demonstrate that WT p97 can unfold proteins and that this activity is dependent on the p97 adaptor NPLOC4-UFD1L, ATP hydrolysis, and substrate ubiquitination, with branched chains providing maximal stimulation. Furthermore, we show that a p97 mutant that causes inclusion body myopathy, Paget's disease of bone, and frontotemporal dementia in humans unfolds substrate faster, suggesting that excess activity may underlie pathogenesis. This work overcomes a significant barrier in the study of p97 and will allow the future dissection of p97 mechanism at a level of detail previously unattainable.

Keywords: AAA ATPase; Cdc48; Npl4; Proteasome; Ufd1.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Adaptor Proteins, Vesicular Transport
  • Adenosine Triphosphate / metabolism
  • Frontotemporal Dementia / etiology
  • Frontotemporal Dementia / genetics*
  • Frontotemporal Dementia / metabolism*
  • Humans
  • Hydrolysis
  • Intracellular Signaling Peptides and Proteins
  • Kinetics
  • Muscular Dystrophies, Limb-Girdle / etiology
  • Muscular Dystrophies, Limb-Girdle / genetics*
  • Muscular Dystrophies, Limb-Girdle / metabolism*
  • Mutation
  • Myositis, Inclusion Body / etiology
  • Myositis, Inclusion Body / genetics*
  • Myositis, Inclusion Body / metabolism*
  • Nuclear Proteins / metabolism*
  • Osteitis Deformans / etiology
  • Osteitis Deformans / genetics*
  • Osteitis Deformans / metabolism*
  • Protein Unfolding
  • Proteins / metabolism*
  • Proteolysis
  • Recombinant Fusion Proteins / chemistry
  • Recombinant Fusion Proteins / genetics
  • Recombinant Fusion Proteins / metabolism
  • Substrate Specificity
  • Ubiquitin / metabolism
  • Valosin Containing Protein / chemistry
  • Valosin Containing Protein / genetics*
  • Valosin Containing Protein / metabolism*

Substances

  • Adaptor Proteins, Vesicular Transport
  • Intracellular Signaling Peptides and Proteins
  • NPLOC4 protein, human
  • Nuclear Proteins
  • Proteins
  • Recombinant Fusion Proteins
  • UFD1 protein, human
  • Ubiquitin
  • Adenosine Triphosphate
  • VCP protein, human
  • Valosin Containing Protein

Supplementary concepts

  • Inclusion Body Myopathy With Early-Onset Paget Disease And Frontotemporal Dementia